• Journal of Animal Science and Technology
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• v.55 no.1
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• pp.41-49
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• 2013

In vitro and in situ incubation studies were conducted to determine effects of yeast culture supplements (Saccharomyces cerevisiae) on cellulolytic bacterial function and fiber digestion in rice straw. In vitro dry matter digestibility of rice straw gradually increased according to supplemental levels of yeast culture (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0%). Digestibility of rice straw started to increase apparently when yeast culture was added more than 0.6% level (p<0.05). Also, we reconfirmed that in vitro dry matter digestibility was significantly increased by 0.6% of yeast culture addition in 4% NaOH treated and non-treated rice straws (p<0.05). When in situ dry matter digestibility was tested in Korean native goats fed basal diet or experimental diet which contained 1.0% of yeast culture, the yeast culture feeding improved in situ dry matter digestibility in both 4% NaOH treated and non-treated rice straws (p<0.05). In case of real-time PCR monitoring cellulolytic bacterial function, the bacterial population attached on rice straw showed the increasing trends with higher level of yeast culture spraying on rice straw. F. succinogenes and R. flavefaciens were significantly increased in accordance to spraying levels of yeast culture (0.0, 0.1 and 0.3%) at both 12 and 24 hrs of in situ incubation (p<0.05). R. albus was significantly higher population in yeast culture spraying than non-soraying at 12 hrs of in situ incubation (p<0.05). These bacterial populations were showed the increasing trends with digestibility enhancement of rice straw according to the higher levels of yeast culture supplement. Overall, these results clearly suggest that the presence of yeast culture result in noticeable increase of rice straw digestion, which is modulated via good effect on cellulolytic bacterial attachment to fiber substrates.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.58-58
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• 2001

본 연구는 섬유소 분해효소 유전자 Cel D(Cellulase Digestion)가 도입된 형질전환 돼지 생산을 통하여 섬유소 함유 사료의 이용 효율을 증대시키고, 나아가 장기이식동물 및 고가의 의료용 단백질 생산가축을 개발하기 위한 원천기술 확보에 있다. 섬유소분해 유전자의 크로닝 및 조직 특이적 발현벡터를 개발하기 위하여, 우선 소의 위중 제4위 내의 미생물로부터 전체 유전자를 분리하였고, 이렇게 작성된 DNA library에서 섬유소분해 관련 유전자인 약 2.0 kb의 Cel D유전자를 크로닝하였으며, 췌장 특이적 발현 프로모터(rat elastase I: 약 200bp)를 크로닝한 후, 미세주입용 형질전환 재조합 벡터를 구축하기 위하여 rElastase I 프로모터 하류에 섬유소 분해 유전자(Cel D)를 연결하여 약 3.0 kb 크기의 재조합 벡터를 준비하였으며, 재조합 유전자를 1세포기 수정란 전핵내에 미세주입 하기 위해 Sal I과 BglII를 이용 유전자 단편을 만들었다. 구축된 유전자를 미세주입하기 위한 수정란을 회수하기위해 총68두의 돼지를 4-5두씩 분리사육하면서 발정동기화 및 과배란 유기를 위해 PG600, Altrenogest, FSH, hCG를 투여하였으며 hCG투여후 약54시간에 외과적 방법에 의해 총 1,359개의 수정란을 회수하였고, 이중 미세주입가능한 1세포기 수정란은 1,296개로 두당 평균 15.9개 였다. 1,296개의 1세포기 수정란 중에서 재조합 유전자(rE I-CelD)가 미세주입된 660개의 수정란을 32두의 수란돈에 외과적 방법에 의해 이식하였으며, 이식되어진 모돈 13두가 분만하여 40.6%의 임신율을 나타내었다. 이렇게 분만된 13두에서 총 65두(암:33두, 수:32두)의 자돈이 생산되었으며, 형질전환 여부를 판명하기 위해 자돈의 꼬리조직으로 부터 genomic DNA를 추출하고 PCR 검정을 실시하였다. PCR 검정 결과, 섬유소 분해 유전자가 도입된 자돈은 5두 이었으며, 그 결과를 Table에 나타내었다.(Table Omitted) Table 1 에서와 같이 섬유소 분해효소유전자가 형질전환된 자돈은 65마리 중 5마리로 7.69%의 형질전환율을 나타내었으며, 5마리의 자돈중 2두(암:1두, 수:1두)는 분만 후 즉시 폐사되었으며 2두(암:1두, 수:1두)는 86일령 그리고 14일령에 폐사하여 현재 1두(암)가 생존하여 섬유소 분해 사양 실험 중에 있다.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.81-86
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• 2005

One of the endoglucanases, F-I-III, was purified from the culture filtrate of T. sp. C-4 through procedures including chromatography on Sephacryl S-200, DEAE-Sepharose A-50, and Chromatofocusing on Mono-P (FPLC). The molecular weight of the enzyme was determined to be about 56,000 Da by SDS-PAGE, and pI of 4.9 by analytical isoelectric focusing. F-I-III showed the highest enzyme activity at $55^{\circ}C$, and the pH optimum of the enzyme was 5.0. There was no loss of activity when the enzyme was incubated at $50^{\circ}C$ for 24 hours. The specific activity of the enzyme F-I-III toward the CMC was 315.4 U/mg. The Km value for $PNPG_2$ of F-I-III was 2.69 mM. N-terminal sequence of F-I-III was analyzed to be QPGTSTPEVHPKKLTTYK. It showed $95\%$ of homology to that of EGI from T. reesei. The presence of some metal ions (1 mM) had only a little effect on CMCase activity. The treatment of the reducing agents resulted in the increase of endoglucanase activity.

• Microbiology and Biotechnology Letters
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• v.8 no.1
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• pp.41-46
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• 1980

Low molecular weight endo-glucanase was partially purified from cellulase complex using Sephadex G-100 gel chromatography. Biochemical properties of the purified component was investigated. Optimum pH and temperature determined were 6.0 and 5$0^{\circ}C$, respectively. Enzymatic hydrolysis of four cellulosic substrates having varying crystallinity was evaluated. It was found that hydrolysis of amorphous region was followed by the hydrolysis of crystalline region. In order to examine the effect of adsorption of the enzyme onto the cellulosic substrates on the hydrolysis kinetics, adsorption studies were carried out. Time course of adsorption of low molecular weight endo-glucanase onto various cellulostances was observed for 25 min. The rate and amount of adsorption to amorphous cellulose was greater than those to the crystalline cellulose. This result suggested that the role of endo-glucanasc was more important to the hydrolysis of amorphous cellulose than to the crystalline region of the cellulose.

• Korean Journal of Microbiology
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• v.6 no.3
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• pp.87-91
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• 1968

To research the characteristics of cellulose produced from one strains of Rhizopus(R-B 14) when it acts on the carboxyl methyl cellulose, this experiment was carried out in crude purified cellulose. The results obtained were as follows: 1. The optimum pH of cellulose was from 4. 5 to 5 .5 and the range of its stability to the pH was considerably extensive and it was from 4 to 7. 2. The optimum temperature of cellulose was $50^{\circ}C$ and the activity of it was instantly inactivated at $70^{\circ}C$. 3. The inhibition of the metal ions to the activity of cellulose was as follows; $Hg^{++}$ > $Ag^+$ +/ > $Fe^{+++}$ > $Pb^{++}$. But the activity of cellulose was not affected at all by $Na^+$, $K^+$ and $Mg^{++}$.

• Korean Journal of Microbiology
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• v.22 no.3
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• pp.143-150
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• 1984

The effect of growth inhibitors on colonial growth of cellulolytic fungus, genus Trichoderma, was investigated to develop a method for the effective screening of various auxotrophs and hypercellulolytic strains. As the results, it was revealed that non-ionic detergents such as Sodium deoxycholate and Triton X-100 were the better ones as a growth inhibitor than Oxgall which has been used to restrict the colony size in genus Trichoderma. Each individual colony remained distinct on minimal plate supplemented with 0.05% Triton X-100 for as long as two or more weeks. The screening of 150 to 200 colonies simultaneous on a single plate was possible in the presence of Triton X-100. The effect of Triton X-100 on simultaneous screening was higher than that of Oxgall by a factor of two.

• Journal of Animal Science and Technology
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• v.49 no.5
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• pp.667-676
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• 2007

This study was conducted to determine effects of cellulolytic microbes inoculation to sawdust-based spent mushroom substrate(SMS) during deepstacking on fermentation parameters, total microbial counts and cellulolytic enzyme activity and to on SMS nutrients utilization by sheep. For sheep metabolism trials, six sheep(ram, average 54.8kg) were fed a Control diet(70% concentrates, 15% rice straw and 15% SMS with no microbial treatment on a dry basis) and a Treatment diet(the same diet including SMS with a microbial treatment) for 2 trials. Spent mushroom substrates with or without a microbial(4 strains including 1 strain of Enterobacter ludwigii, 1 strain of Bacillus cereus and 2 strains of Bacillus subtillis) treatment (1% of SMS on wet basis) were deepstacked for 7 days. The internal temperatures in 1.2 M/T of SMS deepstacks reached to 50±5℃ within 7 days of storage. Total microbial counts remarkably decreased (P<0.05) with a deepstacking process and were not affected(P>0.05) by the microbial treatment. For fibrolytic enzyme activity, CMCase and xylanase activities were decreased(P<0.05) by a deepstacking process. After deepstacking, the microbial treatment showed about 2.5-times higher(P<0.05) for CMCase activity and about 4-times higher(P<0.05) for xylanase activity than those of the Control. Activities of ligninolytic enzymes such as laccase and MnP were not affected by the microbial treatment. The sheep fed the microbially treated SMS diet had a tendency of greater total tract digestibilities of ash(P=0.051), NFE (P=0.071), hemicellulose(P=0.087) and NDF(P=0.096) than those fed the untreated SMS diet. Nitrogen balance of sheep was not affected(P>0.05) by feeding of microbially treated SMS. Accordingly, these results indicate that cellulolytic microbes inoculation during deepstacking of SMS may improve the bio- utilization of SMS by sheep.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.322-325
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• 2005

Pseudomonas sp. JH1014 was isolated from stream water as a detergent-compatible alkaline protease producing microorganism. The strain produced no detectable cellulolytic activity in LB medium. The addition of carboxymethyl cellulose induced the production of carboxymethyl cellulase (CMCase) without causing any significant change in the growth pattern of the strain. The strain reached its maximum growth after 9 to 12 h at $37^{\circ}C$, and the production of CMCase in the presence of the substrate reached its maximum after 21 h of growth at $37^{\circ}C$. The optimum pH of the crude enzyme preparation was pH 6.0. The enzyme had an optimal temperature at $55^{\circ}C$, and retained 70% of its original activity when preincubated at $70^{\circ}C$ for 10 min. Activity staining of the crude enzyme preparation separated on an SDS-PAGE gel showed two active bands with molecular masses of 54 and 30 kDa, indicating that Pseudomonas sp. JH1014 produced at least 2 kinds of CMCase.

• Korean Journal of Microbiology
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• v.14 no.2
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• pp.84-94
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• 1976

Cellulase components, CMCase(Cx) and Avicelase$(C_1)$, were partially prueified, from the culture extract of a strain of Trichoderma koningii by column chromatography on DEAE-Sephadex A-50 and the step of gel filtration through Sphadex G-150, Optimum pH of CMCase was 5.2 and Avicelase showed the highest activity at pH 5.6 in acetate buffer. Optimal temperatures for activities of CMCase and Avicelase were $50^{\circ}C\;and\;60^{\circ}C, $ respectively. More than 70% of the activities of two enzymes were remained after heating at $60^{\circ}C$ for 30 min and Avicelase is more stable than any other fungal enzymes. The Michaelis constants, Km, of CMCase and Avicelase were 0.116% of CMC and 0.281% of avicel. And also the values of maximum velocity, Vmax, of CMCase and Avicelase were $23.20{\mu}g\;and\;2.54{\mu}g$ of reducing sugar per min. Of the metal ions tested against the activites of CMCase and Avicelase, $Cu^{++}, \; Hg^{++}, \;and\;Pb^{{++}$ are remarkably effective inhibitors. The molecular weights of Cx and $C_1$ component were estimated to be about 47, 000 and 61, 000 by gel filtration method.

• Korean Journal of Microbiology
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• v.14 no.1
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• pp.17-24
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• 1976

Celluloytic microorgnasims were isolated form the various sources and four of them were identified as Trichoderma roningi, Aspergillus niger, Penicillium chrysogenum, and Streptomyces sp. The induction of extracellular5 cellulase of these species in the liquid culture media containing carboxymethylcellulose (CMC) or Avicel as inducer showed that CMC was a better effective inducer for the production of CMCase(Cx cellulase component) as well as Avicelase(C$_{1}$ cellulase component) than Avicel. It is believed that certain hydrolysis products of cellulose(CMC) could serve as an inducer for an enzyme synthesis. In T. roningi, Asp. niger, and Strptomyces sp., the optimum temperature of CNCase on CMC-culture medium was 50.deg. but temperature around 40.deg.C was found to be optimum for the activities of CMCase prepared from P.ehrysogenum. The optimum temperature for Avicelase activitiles on Avicel-culture media of T. roningi and P. chrysogenum was $40{\circ}C$ whereas temperature $50{\circ}C$ was found to be optimum for Avicelase from A.niger and Streptomyces sp. The optimal activities of these CNCase and Avicelase prepared from. T. ronigi, Pen.chrysogenum and Streptomyces sp. were found similarly to be at pH's around 5.4 and 6.0 while pH 4.8 was optimum for the activities of CMCase and Avicelase from A.niger, indicating that A.niger in acidic media would yield an enzyme of high activity.